Array of oligonucleotides on a solid substrate

ABSTRACT

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Rule 60 Division of U.S. application Ser. No.850,356, filed Mar. 12, 1992, which is a Rule 60 Division of U.S.application Ser. No. 492,462, filed Mar. 7, 1990, now U.S. Pat. No.5,143,854, which is a Continuation-in-Part of U.S. application Ser. No.362,901, filed Jun. 7, 1989, now abandoned, all assigned to the assigneeof the present invention.

The file of this patent contains drawings executed in color. Copies ofthis patent with color drawings will be provided by the Patent andTrademark Office upon request and payment of the necessary fee.

COPYRIGHT NOTICE

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND OF THE INVENTION

The present inventions relate to the synthesis and placement ofmaterials at known locations. In particular, one embodiment of theinventions provides a method and associated apparatus for preparingdiverse chemical sequences at known locations on a single substratesurface. The inventions may be applied, for example, in the field ofpreparation of oligomer, peptide, nucleic acid, oligosaccharide,phospholipid, polymer, or drug congener preparation, especially tocreate sources of chemical diversity for use in screening for biologicalactivity.

The relationship between structure and activity of molecules is afundamental issue in the study of biological systems. Structure-activityrelationships are important in understanding, for example, the functionof enzymes, the ways in which cells communicate with each other, as wellas cellular control and feedback systems.

Certain macromolecules are known to interact and bind to other moleculeshaving a very specific three-dimensional spatial and electronicdistribution. Any large molecule having such specificity can beconsidered a receptor, whether it is an enzyme catalyzing hydrolysis ofa metabolic intermediate, a cell-surface protein mediating membranetransport of ions, a glycoprotein serving to identify a particular cellto its neighbors, an IgG-class antibody circulating in the plasma, anoligonucleotide sequence of DNA in the nucleus, or the like. The variousmolecules which receptors selectively bind are known as ligands.

Many assays are available for measuring the binding affinity of knownreceptors and ligands, but the information which can be gained from suchexperiments is often limited by the number and type of ligands which areavailable. Novel ligands are sometimes discovered by chance or byapplication of new techniques for the elucidation of molecularstructure, including x-ray crystallographic analysis and recombinantgenetic techniques for proteins.

Small peptides are an exemplary system for exploring the relationshipbetween structure and function in biology. A peptide is a sequence ofamino acids. When the twenty naturally occurring amino acids arecondensed into polymeric molecules they form a wide variety ofthree-dimensional configurations, each resulting from a particular aminoacid sequence and solvent condition. The number of possiblepentapeptides of the 20 naturally occurring amino acids, for example, is20⁵ or 3.2 million different peptides. The likelihood that molecules ofthis size might be useful in receptor-binding studies is supported byepitope analysis studies showing that some antibodies recognizesequences as short as a few amino acids with high specificity.Furthermore, the average molecular weight of amino acids puts smallpeptides in the size range of many currently useful pharmaceuticalproducts.

Pharmaceutical drug discovery is one type of research which relies onsuch a study of structure-activity relationships. In most cases,contemporary pharmaceutical research can be described as the process ofdiscovering novel ligands with desirable patterns of specificity forbiologically important receptors. Another example is research todiscover new compounds for use in agriculture, such as pesticides andherbicides.

Sometimes, the solution to a rational process of designing ligands isdifficult or unyielding. Prior methods of preparing large numbers ofdifferent polymers have been painstakingly slow when used at a scalesufficient to permit effective rational or random screening. Forexample, the "Merrifield" method (J. Am. Chem. Soc. (1963) 85:2149-2154,which is incorporated herein by reference for all purposes) has beenused to synthesize peptides on a solid support. In the Merrifieldmethod, an amino acid is covalently bonded to a support made of aninsoluble polymer. Another amino acid with an alpha protected group isreacted with the covalently bonded amino acid to form a dipeptide. Afterwashing, the protective group is removed and a third amino acid with analpha protective group is added to the dipeptide. This process iscontinued until a peptide of a desired length and sequence is obtained.Using the Merrifield method, it is not economically practical tosynthesize more than a handful of peptide sequences in a day.

To synthesize larger numbers of polymer sequences, it has also beenproposed to use a series of reaction vessels for polymer synthesis. Forexample, a tubular reactor system may be used to synthesize a linearpolymer on a solid phase support by automated sequential addition ofreagents. This method still does not enable the synthesis of asufficiently large number of polymer sequences for effective economicalscreening.

Methods of preparing a plurality of polymer sequences are also known inwhich a porous container encloses a known quantity of reactiveparticles, the particles being larger in size than pores of thecontainer. The containers may be selectively reacted with desiredmaterials to synthesize desired sequences of product molecules. As withother methods known in the art, this method cannot practically be usedto synthesize a sufficient variety of polypeptides for effectivescreening.

Other techniques have also been described. These methods include thesynthesis of peptides on 96 plastic pins which fit the format ofstandard microtiter plates. Unfortunately, while these techniques havebeen somewhat useful, substantial problems remain. For example, thesemethods continue to be limited in the diversity of sequences which canbe economically synthesized and screened.

From the above, it is seen that an improved method and apparatus forsynthesizing a variety of chemical sequences at known locations isdesired.

SUMMARY OF THE INVENTION

An improved method and apparatus for the preparation of a variety ofpolymers is disclosed.

In one preferred embodiment, linker molecules are provided on asubstrate. A terminal end of the linker molecules is provided with areactive functional group protected with a photoremovable protectivegroup. Using lithographic methods, the photoremovable protective groupis exposed to light and removed from the linker molecules in firstselected regions. The substrate is then washed or otherwise contactedwith a first monomer that reacts with exposed functional groups on thelinker molecules. In a preferred embodiment, the monomer is an aminoacid containing a photoremovable protective group at its amino orcarboxy terminus and the linker molecule terminates in an amino orcarboxy acid group bearing a photoremovable protective group.

A second set of selected regions is, thereafter, exposed to light andthe photoremovable protective group on the linker molecule/protectedamino acid is removed at the second set of regions. The substrate isthen contacted with a second monomer containing a photoremovableprotective group for reaction with exposed functional groups. Thisprocess is repeated to selectively apply monomers until polymers of adesired length and desired chemical sequence are obtained. Photolabilegroups are then optionally removed and the sequence is, thereafter,optionally capped. Side chain protective groups, if present, are alsoremoved.

By using the lithographic techniques disclosed herein, it is possible todirect light to relatively small and precisely known locations on thesubstrate. It is, therefore, possible to synthesize polymers of a knownchemical sequence at known locations on the substrate.

The resulting substrate will have a variety of uses including, forexample, screening large numbers of polymers for biological activity. Toscreen for biological activity, the substrate is exposed to one or morereceptors such as antibodies whole cells, receptors on vesicles, lipids,or any one of a variety of other receptors. The receptors are preferablylabeled with, for example, a fluorescent marker, radioactive marker, ora labeled antibody reactive with the receptor. The location of themarker on the substrate is detected with, for example, photon detectionor autoradiographic techniques. Through knowledge of the sequence of thematerial at the location where binding is detected, it is possible toquickly determine which sequence binds with the receptor and, therefore,the technique can be used to screen large numbers of peptides. Otherpossible applications of the inventions herein include diagnostics inwhich various antibodies for particular receptors would be placed on asubstrate and, for example, blood sera would be screened for immunedeficiencies. Still further applications include, for example, selective"doping" of organic materials in semiconductor devices, and the like.

In connection with one aspect of the invention an improved reactorsystem for synthesizing polymers is also disclosed. The reactor systemincludes a substrate mount which engages a substrate around a peripherythereof. The substrate mount provides for a reactor space between thesubstrate and the mount through or into which reaction fluids are pumpedor flowed. A mask is placed on or focused on the substrate andilluminated so as to deprotect selected regions of the substrate in thereactor space. A monomer is pumped through the reactor space orotherwise contacted with the substrate and reacts with the deprotectedregions. By selectively deprotecting regions on the substrate andflowing predetermined monomers through the reactor space, desiredpolymers at known locations may be synthesized.

Improved detection apparatus and methods are also disclosed. Thedetection method and apparatus utilize a substrate having a largevariety of polymer sequences at known locations on a surface thereof.The substrate is exposed to a fluorescently labeled receptor which bindsto one or more of the polymer sequences. The substrate is placed in amicroscope detection apparatus for identification of locations wherebinding takes place. The microscope detection apparatus includes amonochromatic or polychromatic light source for directing light at thesubstrate, means for detecting fluoresced light from the substrate, andmeans for determining a location of the fluoresced light. The means fordetecting light fluoresced on the substrate may in some embodimentsinclude a photon counter. The means for determining a location of thefluoresced light may include an x/y translation table for the substrate.Translation of the slide and data collection are recorded and managed byan appropriately programmed digital computer.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates masking and irradiation of a substrate at a firstlocation. The substrate is shown in cross-section;

FIG. 2 illustrates the substrate after application of a monomer "A";

FIG. 3 illustrates irradiation of the substrate at a second location;

FIG. 4 illustrates the substrate after application of monomer "B";

FIG. 5 illustrates irradiation of the "A" monomer;

FIG. 6 illustrates the substrate after a second application of "B";

FIG. 7 illustrates a completed substrate;

FIGS. 8A and 8B illustrate alternative embodiments of a reactor systemfor forming a plurality of polymers on a substrate;

FIG. 9 illustrates a detection apparatus for locating fluorescentmarkers on the substrate;

FIGS. 10A-10M illustrate the method as it is applied to the productionof the trimers of monomers "A" and "B";

FIGS. 11A and 11B are fluorescence traces for standard fluorescentbeads;

FIGS. 12A and 12B are fluorescence curves for NVOC(6-nitroveratryloxycarbonyl) slides not exposed and exposed to lightrespectively;

FIGS. 13A to 13D are fluorescence plots of slides exposed through 100μm, 50 μm, 20 μm, and 10 μm masks; 14A and 14B illustrate formation ofYGGFL (a peptide of sequenceH2N-tyrosine-glycine-glycine-phenylalanine-leucine-CO₂ H) and GGFL (apeptide of sequence H₂ N-glycine-glycine-phenylalanine-leucine-CO₂ H),followed by exposure to labeled Herz antibody (an antibody thatrecognizes YGGFL but not GGFL);

FIGS. 15A and 15B fluorescence plots of a slide with a checkerboardpattern of YGGFL and GGFL exposed to labeled Herz antibody; FIG. 15Aillustrates a 500×500 μm mask which has been focused on the substrateaccording to FIG. 8A while FIG. 15B illustrates a 50×50 μm mask placedin direct contact with the substrate in accord with FIG. 8B;

FIG. 16 is a fluorescence plot of YGGFL and PGGFL synthesized in a 50 μmcheckerboard pattern;

FIG. 17 is a fluorescence plot of YPGGFL and YGGFL synthesized in a 50μm checkerboard pattern;

FIGS. 18A and 18B illustrate the mapping of sixteen sequencessynthesized on two different glass slides;

FIG. 19 is a fluorescence plot of the slide illustrated in FIG. 18A; and

FIG. 20 is a fluorescence plot of the slide illustrated in FIG. 10B.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS

I. Glossary

II. General

III. Polymer Synthesis

IV. Details of One Embodiment of a Reactor System

V. Details of One Embodiment of a Fluorescent Detection Device

VI. Determination of Relative Binding Strength of Receptors

VII. Examples

A. Slide Preparation

B. Synthesis of Eight Trimers of "A" and "B"

C. Synthesis of a Dimer of an Aminopropyl Group and a Fluorescent Group

D. Demonstration of Signal Capability

E. Determination of the Number of Molecules Per Unit Area

F. Removal of NVOC and Attachment of a Fluorescent Marker

G. Use of a Mask in Removal of NVOC

H. Attachment of YGGFL and Subsequent Exposure to Herz Antibody and GoatAntimouse

I. Monomer-by-Monomer Formation of YGGFL and Subsequent Exposure toLabeled Antibody

J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL

K. Monomer-by Monomer Synthesis of YGGFL and YPGGFL

L. Synthesis of an Array of Sixteen Different Amino Acid Sequences andEstimation of Relative Binding Affinity to Herz Antibody

VIII. Illustrative Alternative Embodiment

IX. Conclusion

I. Glossary

The following terms are intended to have the following general meaningsas they are used herein:

1. Complementary: Refers to the topological compatibility or matchingtogether of interacting surfaces of a ligand molecule and its receptor.Thus, the receptor and its ligand can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other.

2. Epitope: The portion of an antigen molecule which is delineated bythe area of interaction with the subclass of receptors known asantibodies.

3. Ligand: A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,steroids, etc.), hormone receptors, peptides, enzymes, enzymesubstrates, cofactors, drugs (e.g., opiates, etc), lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies.

4. Monomer: A member of the set of small molecules which can be joinedtogether to form a polymer. The set of monomers includes but is notrestricted to, for example, the set of common L-amino acids, the set ofD-amino acids, the set of synthetic amino acids, the set of nucleotidesand the set of pentoses and hexoses. As used herein, monomers refers toany member of a basis set for synthesis of a polymer. For example,dimers of L-amino acids form a basis set of 400 monomers for synthesisof polypeptides. Different basis sets of monomers may be used atsuccessive steps in the synthesis of a polymer.

5. Peptide: A polymer in which the monomers are alpha amino acids andwhich are joined together through amide bonds and alternatively referredto as a polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are more than two amino acid monomers long,and often more than 20 amino acid monomers long. Standard abbreviationsfor amino acids are used (e.g., P for proline). These abbreviations areincluded in Stryer, Biochemstry, Third Ed., 1988, which is incorporatedherein by reference for all purposes.

6. Radiation: Energy which may be selectively applied including energyhaving a wavelength of between 10⁻¹⁴ and 10⁴ meters including, forexample, electron beam radiation, gamma radiation, x-ray radiation,ultraviolet radiation, visible light, infrared radiation, microwaveradiation, and radio waves. "Irradiation" refers to the application ofradiation to a surface.

7. Receptor: A molecule that has an affinity for a given ligand.Receptors may be naturally-occuring or manmade molecules. Also, they canbe employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A"Ligand Receptor Pair" is formed when two macromolecules have combinedthrough molecular recognition to form a complex.

Other examples of receptors which can be investigated by this inventioninclude but are not restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in a new class of antibiotics. Ofparticular value would be antibiotics against opportunistic fungi,protozoa, and those bacteria resistant to the antibiotics in currentuse.

b) Enzymes: For instance, the binding site of enzymes such as theenzymes responsible for cleaving neurotransmitters; determination ofligands which bind to certain receptors to modulate the action of theenzymes which cleave the different neurotransmitters is useful in thedevelopment of drugs which can be used in the treatment of disorders ofneurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for auto immunediseases (e.g., by blocking the binding of the "self" antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant. Catalytic polypeptides are described in, for example, U.S.application Ser. No. 404,920, which is incorporated herein by referencefor all purposes.

f) Hormone receptors: For instance, the receptors for insulin and growthhormone. Determination of the ligands which bind with high affinity to areceptor is useful in the development of, for example, an oralreplacement of the daily injections which diabetics must take to relievethe symptoms of diabetes, and in the other case, a replacement for thescarce human growth hormone which can only be obtained from cadavers orby recombinant DNA technology. Other examples are the vasoconstrictivehormone receptors; determination of those ligands which bind to areceptor may lead to the development of drugs to control blood pressure.

g) Opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

8. Substrate: A material having a rigid or semi-rigid surface. In manyembodiments, at least one surface of the substrate will be substantiallyflat, although in some embodiments it may be desirable to physicallyseparate synthesis regions for different polymers with, for example,wells, raised regions, etched trenches, or the like. According to otherembodiments, small beads may be provided on the surface which may bereleased upon completion of the synthesis.

9. Protective Group: A material which is bound to a monomer unit andwhich may be spatially removed upon selective exposure to an activatorsuch as electromagnetic radiation. Examples of protective groups withutility herein include Nitroveratryloxy carbonyl, Nitrobenzyloxycarbonyl, Dimethyl dimethoxybenzyloxy carbonyl,5-Bromo-7-nitroindolinyl, o-Hydroxy-α-methyl cinnamoyl, and2-Oxymethylene anthraquinone. Other examples of activators include ionbeams, electric fields, magnetic fields, electron beams, x-ray, and thelike.

10. Predefined Region: A predefined region is a localized area on asurface which is, was, or is intended to be activated for formation of apolymer. The predefined region may have any convenient shape, e.g.,circular, rectangular, elliptical, wedge-shaped, etc. For the sake ofbrevity herein, "predefined regions" are sometimes referred to simply as"regions."

11. Substantially Pure: A polymer is considered to be "substantiallypure" within a predefined region of a substrate when it exhibitscharacteristics that distinguish it from other predefined regions.Typically, purity will be measured in terms of biological activity orfunction as a result of uniform sequence. Such characteristics willtypically be measured by way of binding with a selected ligand orreceptor.

II. General

The present invention provides methods and apparatus for the preparationand use of a substrate having a plurality of polymer sequences inpredefined regions. The invention is described herein primarily withregard to the preparation of molecules containing sequences of aminoacids, but could readily be applied in the preparation of otherpolymers. Such polymers include, for example, both linear and cyclicpolymers of nucleic acids, polysaccharides, phospholipids, and peptideshaving either α-, β-, or ω-amino acids, heteropolymers in which a knowndrug is covalently bound to any of the above, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, or other polymerswhich will be apparent upon review of this disclosure. In a preferredembodiment, the invention herein is used in the synthesis of peptides.

The prepared substrate may, for example, be used in screening a varietyof polymers as ligands for binding with a receptor, although it will beapparent that the invention could be used for the synthesis of areceptor for binding with a ligand. The substrate disclosed herein willhave a wide variety of other uses. Merely by way of example, theinvention herein can be used in determining peptide and nucleic acidsequences which bind to proteins, finding sequence-specific bindingdrugs, identifying epitopes recognized by antibodies, and evaluation ofa variety of drugs for clinical and diagnostic applications, as well ascombinations of the above.

The invention preferably provides for the use of a substrate "S" with asurface. Linker molecules "L" are optionally provided on a surface ofthe substrate. The purpose of the linker molecules, in some embodiments,is to facilitate receptor recognition of the synthesized polymers.

Optionally, the linker molecules may be chemically protected for storagepurposes. A chemical storage protective group such as t-BOC(t-butoxycarbonyl) may be used in some embodiments. Such chemicalprotective groups would be chemically removed upon exposure to, forexample, acidic solution and would serve to protect the surface duringstorage and be removed prior to polymer preparation.

On the substrate or a distal end of the linker molecules, a functionalgroup with a protective group P₀ is provided. The protective group P₀may be removed upon exposure to radiation, electric fields, electriccurrents, or other activators to expose the functional group.

In a preferred embodiment, the radiation is ultraviolet (UV), infrared(IR), or visible light. As more fully described below, the protectivegroup may alternatively be an electrochemically-sensitive group whichmay be removed in the presence of an electric field. In still furtheralternative embodiments, ion beams, electron beams, or the like may beused for deprotection.

In some embodiments, the exposed regions and, therefore, the area uponwhich each distinct polymer sequence is synthesized are smaller thanabout 1 cm² or less than 1 mm². In preferred embodiments the exposedarea is less than about 10,000 μm² or, more preferably, less than 100μm² and may, in some embodiments, encompass the binding site for as fewas a single molecule. Within these regions, each polymer is preferablysynthesized in a substantially pure form.

Concurrently or after exposure of a known region of the substrate tolight, the surface is contacted with a first monomer unit M₁ whichreacts with the functional group which has been exposed by thedeprotection step. The first monomer includes a protective group P₁. P₁may or may not be the same as P₀.

Accordingly, after a first cycle, known first regions of the surface maycomprise the sequence:

    S-L-M.sub.1 -P.sub.1

while remaining regions of the surface comprise the sequence:

    S-L-P.sub.0.

Thereafter, second regions of the surface (which may include the firstregion) are exposed to light and contacted with a second monomer M₂(which may or may not be the same as M₁) having a protective group P₂.P₂ may or may not be the same as P₀ and P₁. After this second cycle,different regions of the substrate may comprise one or more of thefollowing sequences:

    S-L-M.sub.1 -M.sub.2 -P.sub.2 S-L-M.sub.2 -P.sub.2 S-L-M.sub.1 -P.sub.1 and/or S-L-P.sub.0.

The above process is repeated until the substrate includes desiredpolymers of desired lengths. By controlling the locations of thesubstrate exposed to light and the reagents exposed to the substratefollowing exposure, the location of each sequence will be known.

Thereafter, the protective groups are removed from some or all of thesubstrate and the sequences are, optionally, capped with a capping unitC. The process results in a substrate having a surface with a pluralityof polymers of the following general formula:

    S-[L]-(M.sub.i)-(M.sub.j)-(M.sub.k) . . . (M.sub.x)-[C]

where square brackets indicate optional groups, and M_(i) . . . M_(x)indicates any sequence of monomers. The number of monomers could cover awide variety of values, but in a preferred embodiment they will rangefrom 2 to 100.

In some embodiments a plurality of locations on the substrate polymersare to contain a common monomer subsequence. For example, it may bedesired to synthesize a sequence S-M₁ -M₂ -M₃ at first locations and asequence S-M₄ -M₂ -M₃ at second locations. The process would commencewith irradiation of the first locations followed by contacting with M₁-P, resulting in the sequence S-M₁ -P at the first location. The secondlocations would then be irradiated and contacted with M₄ -P, resultingin the sequence S-M₄ -P at the second locations. Thereafter both thefirst and second locations would be irradiated and contacted with thedimer M₂ -M₃, resulting in the sequence S-M₁ -M₂ -M₃ at the firstlocations and S-M₄ -M₂ -M₃ at the second locations. Of course, commonsubsequences of any length could be utilized including those in a rangeof 2 or more monomers, 2 to 100 monomers, 2 to 20 monomers, and a mostpreferred range of 2 to 3 monomers.

According to other embodiments, a set of masks is used for the firstmonomer layer and, thereafter, varied light wavelengths are used forselective deprotection. For example, in the process discussed above,first regions are first exposed through a mask and reacted with a firstmonomer having a first protective group P₁, which is removable uponexposure to a first wavelength of light (e.g., IR). Second regions aremasked and reacted with a second monomer having a second protecive groupP₂, which is removable upon exposure to a second wavelength of light(e.g., UV). Thereafter, masks become unnecessary in the synthesisbecause the entire substrate may be exposed alternatively to the firstand second wavelengths of light in the deprotection cycle.

The polymers prepared on a substrate according to the above methods willhave a variety of uses including, for example, screening for biologicalactivity. In such screening activities, the substrate containing thesequences is exposed to an unlabeled or labeled receptor such as anantibody, receptor on a cell, phospholipid vesicle, or any one of avariety of other receptors. In one preferred embodiment the polymers areexposed to a first, unlabeled receptor of interest and, thereafter,exposed to a labeled receptor-specific recognition element, which is,for example, an antibody. This process will provide signal amplificationin the detection stage.

The receptor molecules may bind with one or more polymers on thesubstrate. The presence of the labeled receptor and, therefore, thepresence of a sequence which binds with the receptor is detected in apreferred embodiment through the use of autoradiography, detection offluorescence with a charge-coupled device, fluorescence microscopy, orthe like. The sequence of the polymer at the locations where thereceptor binding is detected may be used to determine all or part of asequence which is complementary to the receptor.

Use of the invention herein is illustrated primarily with reference toscreening for biological activity. The invention will, however, findmany other uses. For example, the invention may be used in informationstorage (e.g., on optical disks), production of molecular electronicdevices, production of stationary phases in separation sciences,production of dyes and brightening agents, photography, and inimmobilization of cells, proteins, lectins, nucleic acids,polysaccharides and the like in patterns on a surface via molecularrecognition of specific polymer sequences. By synthesizing the samecompound in adjacent, progressively differing concentrations, a gradientwill be established to control chemotaxis or to develop diagnosticdipsticks which, for example, titrate an antibody against an increasingamount of antigen. By synthesizing several catalyst molecules in closeproximity, more efficient multistep conversions may be achieved by"coordinate immobilization." Coordinate immobilization also may be usedfor electron transfer systems, as well as to provide both structuralintegrity and other desirable properties to materials such aslubrication, wetting, etc.

According to alternative embodiments, molecular biodistribution orpharmacokinetic properties may be examined. For example, to assessresistance to intestinal or serum proteases, polymers may be capped witha fluorescent tag and exposed to biological fluids of interest.

III. Polymer Synthesis

FIG. 1 illustrates one embodiment of the invention disclosed herein inwhich a substrate 2 is shown in cross-section. Essentially, anyconceivable substrate may be employed in the invention. The substratemay be biological, nonbiological, organic, inorganic, or a combinationof any of these, existing as particles, strands, precipitates, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,plates, slides, etc. The substrate may have any convenient shape, suchas a disc, square, sphere, circle, etc. The substrate is preferably flatbut may take on a variety of alternative surface configurations. Forexample, the substrate may contain raised or depressed regions on whichthe synthesis takes place. The substrate and its surface preferably forma rigid support on which to carry out the reactions described herein.The substrate and its surface is also chosen to provide appropriatelight-absorbing characteristics. For instance, the substrate may be apolymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs,GaP, SiO₂, SIN₄, modified silicon, or any one of a wide variety of gelsor polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other substrate materials will be readily apparent to those ofskill in the art upon review of this disclosure. In a preferredembodiment the substrate is flat glass or single-crystal silicon withsurface relief features of less than 10 Å.

According to some embodiments, the surface of the substrate is etchedusing well known techniques to provide for desired surface features. Forexample, by way of the formation of trenches, v-grooves, mesastructures, or the like, the synthesis regions may be more closelyplaced within the focus point of impinging light, be provided withreflective "mirror" structures for maximization of light collection fromfluorescent sources, or the like.

Surfaces on the solid substrate will usually, though not always, becomposed of the same material as the substrate. Thus, the surface may becomposed of any of a wide variety of materials, for example, polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, membranes, or any of the above-listedsubstrate materials. In some embodiments the surface may provide for theuse of caged binding members which are attached firmly to the surface ofthe substrate. Preferably, the surface will contain reactive groups,which could be carboxyl, amino, hydroxyl, or the like. Most preferably,the surface will be optically transparent and will have surface Si--OHfunctionalities, such as are found on silica surfaces.

The surface 4 of the substrate is preferably provided with a layer oflinker molecules 6, although it will be understood that the linkermolecules are not required elements of the invention. The linkermolecules are preferably of sufficient length to permit polymers in acompleted substrate to interact freely with molecules exposed to thesubstrate. The linker molecules should be 6-50 atoms long to providesufficient exposure. The linker molecules may be, for example, arylacetylene, ethylene glycol oligomers containing 2-10 monomer units,diamines, diacids, amino acids, or combinations thereof. Other linkermolecules may be used in light of this disclsoure.

According to alternative embodiments, the linker molecules are selectedbased upon their hydrophilic/hydrophobic properties to improvepresentation of synthesized polymers to certain receptors. For example,in the case of a hydrophilic receptor, hydrophilic linker molecules willbe preferred so as to permit the receptor to more closely approach thesynthesized polymer.

According to another alternative embodiment, linker molecules are alsoprovided with a photocleavable group at an intermediate position. Thephotocleavable group is preferably cleavable at a wavelength differentfrom the protective group. This enables removal of the various polymersfollowing completion of the synthesis by way of exposure to thedifferent wavelengths of light.

The linker molecules can be attached to the substrate via carbon-carbonbonds using, for example, (poly)trifluorochloroethylene surfaces, orpreferably, by siloxane bonds (using, for example, glass or siliconoxide surfaces). Siloxane bonds with the surface of the substrate may beformed in one embodiment via reactions of linker molecules bearingtrichlorosilyl groups. The linker molecules may optionally be attachedin an ordered array, i.e., as parts of the head groups in a polymerizedLangmuir Blodgett film. In alternative embodiments, the linker moleculesare adsorbed to the surface of the substrate.

The linker molecules and monomers used herein are provided with afunctional group to which is bound a protective group. Preferably, theprotective group is on the distal or terminal end of the linker moleculeopposite the substrate. The protective group may be either a negativeprotective group (i.e., the protective group renders the linkermolecules less reactive with a monomer upon exposure) or a positiveprotective group (i.e., the protective group renders the linkermolecules more reactive with a monomer upon exposure). In the case ofnegative protective groups an additional step of reactivation will berequired. In some embodiments, this will be done by heating.

The protective group on the linker molecules may be selected from a widevariety of positive light-reactive groups preferably including nitroaromatic compounds such as o-nitrobenzyl derivatives or benzylsulfonyl.In a preferred embodiment, 6-nitroveratryloxycarbonyl (NVOC),2-nitrobenzyloxycarbonyl (NBOC) orα,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ) is used. In oneembodiment, a nitro aromatic compound containing a benzylic hydrogenortho to the nitro group is used, i.e., a chemical of the form: ##STR1##where R₁ is alkoxy, alkyl, halo, aryl, alkenyl, or hydrogen; R₂ isalkoxy, alkyl, halo, aryl, nitro, or hydrogen; R₃ is alkoxy, alkyl,halo, nitro, aryl, or hydrogen; R₄ is alkoxy, alkyl, hydrogen, aryl,halo, or nitro; and R₅ is alkyl, alkynyl, cyano, alkoxy, hydrogen, halo,aryl, or alkenyl. Other materials which may be used includeo-hydroxy-α-methyl cinnamoyl derivatives. Photoremovable protectivegroups are described in, for example, Patchornik, J. Am. Chem. Soc.(1970) 92:6333 and Amit et al., J. Org. Chem. (1974) 39:192, both ofwhich are incorporated herein by reference.

In an alternative embodiment the positive reactive group is activatedfor reaction with reagents in solution. For example, a 5-bromo-7-nitroindoline group, when bound to a carbonyl, undergoes reaction uponexposure to light at 420 nm.

In a second alternative embodiment, the reactive group on the linkermolecule is selected from a wide variety of negative light-reactivegroups including a cinammate group.

Alternatively, the reactive group is activated or deactivated byelectron beam lithography, x-ray lithography, or any other radiation.Suitable reactive groups for electron beam lithography include sulfonyl.Other methods may be used including, for example, exposure to a currentsource. Other reactive groups and methods of activation may be used inlight of this disclosure.

As shown in FIG. 1, the linking molecules are preferably exposed to, forexample, light through a suitable mask 8 using photolithographictechniques of the type known in the semiconductor industry and describedin, for example, Sze, VLSI Technology, McGraw-Hill (1983), and Mead etal., Introduction to VLSI Systems, Addison-Wesley (1980), which areincorporated herein by reference for all purposes. The light may bedirected at either the surface containing the protective groups or atthe back of the substrate, so long as the substrate is transparent tothe wavelength of light needed for removal of the protective groups. Inthe embodiment shown in FIG. 1, light is directed at the surface of thesubstrate containing the protective groups. FIG. 1 illustrates the useof such masking techniques as they are applied to a positive reactivegroup so as to activate linking molecules and expose functional groupsin areas 10a and 10b.

The mask 8 is in one embodiment a transparent support materialselectively coated with a layer of opaque material. Portions of theopaque material are removed, leaving opaque material in the precisepattern desired on the substrate surface. The mask is brought into closeproximity with, imaged on, or brought directly into contact with thesubstrate surface as shown in FIG. 1. "Openings" in the mask correspondto locations on the substrate where it is desired to removephotoremovable protective groups from the substrate. Alignment may beperformed using conventional alignment techniques in which alignmentmarks (not shown) are used to accurately overlay successive masks withprevious patterning steps, or more sophisticated techniques may be used.For example, interferometric techniques such as the one described inFlanders et al., "A New Interferometric Alignment Technique," App. Phys.Lett. (1977) 31:426-428, which is incorporated herein by reference, maybe used.

To enhance contrast of light applied to the substrate, it is desirableto provide contrast enhancement materials between the mask and thesubstrate according to some embodiments. This contrast enhancement layermay comprise a molecule which is decomposed by light such as quinonediazide or a material which is transiently bleached at the wavelength ofinterest. Transient bleaching of materials will allow greaterpenetration where light is applied, thereby enhancing contrast.Alternatively, contrast enhancement may be provided by way of a claddedfiber optic bundle.

The light may be from a conventional incandescent source, a laser, alaser diode, or the like. If non-collimated sources of light are used itmay be desirable to provide a thick- or multi-layered mask to preventspreading of the light onto the substrate. It may, further, be desirablein some embodiments to utilize groups which are sensitive to differentwavelengths to control synthesis. For example, by using groups which aresensitive to different wavelengths, it is possible to select branchpositions in the synthesis of a polymer or eliminate certain maskingsteps. Several reactive groups along with their correspondingwavelengths for deprotection are provided in Table 1.

                  TABLE 1                                                         ______________________________________                                                            Approximate                                               Group               Deprotection Wavelength                                   ______________________________________                                        Nitroveratryloxy carbonyl (NVOC)                                                                  UV (300-400 nm)                                           Nitrobenzyloxy carbonyl (NBOC)                                                                    UV (300-350 nm)                                           Dimethyl dimethoxybenzyloxy                                                                       UV (280-300 nm)                                           carbonyl                                                                      5-Bromo-7-nitroindolinyl                                                                          UV (420 nm)                                               o-Hydroxy-α-methyl cinnamoyl                                                                UV (300-350 nm)                                           2-Oxymethylene anthraquinone                                                                      UV (350 nm)                                               ______________________________________                                    

While the invention is illustrated primarily herein by way of the use ofa mask to illuminate selected regions the substrate, other techniquesmay also be used. For example, the substrate may be translated under amodulated laser or diode light source. Such techniques are discussed in,for example, U.S. Pat. No. 4,719,615 (Feyrer et al.), which isincorporated herein by reference. In alternative embodiments a lasergalvanometric scanner is utilized. In other embodiments, the synthesismay take place on or in contact with a conventional liquid crystal(referred to herein as a "light valve") or fiber optic light sources. Byappropriately modulating liquid crystals, light may be selectivelycontrolled so as to permit light to contact selected regions of thesubstrate. Alternatively, synthesis may take place on the end of aseries of optical fibers to which light is selectively applied. Othermeans of controlling the location of light exposure will be apparent tothose of skill in the art.

The substrate may be irradiated either in contact or not in contact witha solution (not shown) and is, preferably, irradiated in contact with asolution. The solution contains reagents to prevent the by-productsformed by irradiation from interfering with synthesis of the polymeraccording to some embodiments. Such by-products might include, forexample, carbon dioxide, nitrosocarbonyl compounds, styrene derivatives,indole derivatives, and products of their photochemical reactions.Alternatively, the solution may contain reagents used to match the indexof refraction of the substrate. Reagents added to the solution mayfurther include, for example, acidic or basic buffers, thiols,substituted hydrazines and hydroxylamines, reducing agents (e.g., NADH)or reagents known to react with a given functional group (e.g., arylnitroso+glyoxylic acid→aryl formhydroxamate+CO₂).

Either concurrently with or after the irradiation step, the linkermolecules are washed or otherwise contacted with a first monomer,illustrated by "A" in regions 12a and 12b in FIG. 2. The first monomerreacts with the activated functional groups of the linkage moleculeswhich have been exposed to light. The first monomer, which is preferablyan amino acid, is also provided with a photoprotective group. Thephotoprotective group on the monomer may be the same as or differentthan the protective group used in the linkage molecules, and may beselected from any of the above-described protective groups. In oneembodiment, the protective groups for the A monomer is selected from thegroup NBOC and NVOC.

As shown in FIG. 3, the process of irradiating is thereafter repeated,with a mask repositioned so as to remove linkage protective groups andexpose functional groups in regions 14a and 14b which are illustrated asbeing regions which were protected in the previous masking step. As analternative to repositioning of the first mask, in many embodiments asecond mask will be utilized. In other alternative embodiments, somesteps may provide for illuminating a common region in successive steps.As shown in FIG. 3, it may be desirable to provide separation betweenirradiated regions. For example, separation of about 1-5 μm may beappropriate to account for alignment tolerances.

As shown in FIG. 4, the substrate is then exposed to a second protectedmonomer "B," producing B regions 16a and 16b. Thereafter, the substrateis again masked so as to remove the protective groups and exposereactive groups on A region 12a and B region 16b. The substrate is againexposed to monomer B, resulting in the formation of the structure shownin FIG. 6. The dimers B-A and B-B have been produced on the substrate.

A subsequent series of masking and contacting steps similar to thosedescribed above with A (not shown) provides the structure shown in FIG.7. The process provides all possible dimers of B and A, i.e., B-A, A-B,A-A, and B-B.

The substrate, the area of synthesis, and the area for synthesis of eachindividual polymer could be of any size or shape. For example, squares,ellipsoids, rectangles, triangles, circles, or portions thereof, alongwith irregular geometric shapes, may be utilized. Duplicate synthesisareas may also be applied to a single substrate for purposes ofredundancy.

In one embodiment the regions 12a, 12b and 16a, 16b on the substratewill have a surface area of between about 1 cm² and 10⁻¹⁰ cm². In someembodiments the regions 12a, 12b and 16a, 16b have areas of less thanabout 10⁻¹ cm², 10⁻² cm², 10⁻³ cm², 10⁻⁴ cm², 10⁻⁵ cm², 10⁻⁶ cm², 10⁻⁷cm², 10⁻⁸ cm², or 10⁻¹⁰ cm². In a preferred embodiment, the regions 12a,12b and 16a, 16b are between about 10×10 μm and 500×500 μm.

In some embodiments a single substrate supports more than about 10different monomer sequences and perferably more than about 100 differentmonomer sequences, although in some embodiments more than about 10³,10⁴, 10⁵, 10⁶, 10⁷, or 10⁸ different sequences are provided on asubstrate. Of course, within a region of the substrate in which amonomer sequence is synthesized, it is preferred that the monomersequence be substantially pure. In some embodiments, regions of thesubstrate contain polymer sequences which are at least about 1%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97% 98% or 99% pure.

According to some embodiments, several sequences are intentionallyprovided within a single region so as to provide an initial screeningfor biological activity, after which materials within regions exhibitingsignificant binding are further evaluated.

IV. Details of One Embodiment of a Reactor System

FIG. 8A schematically illustrates a preferred embodiment of a reactorsystem 100 for synthesizing polymers on the prepared substrate inaccordance with one aspect of the invention. The reactor system includesa body 102 with a cavity 104 on a surface thereof. In preferredembodiments the cavity 104 is between about 50 and 1000 μm deep with adepth of about 500 μm preferred.

The bottom of the cavity is preferably provided with an array of ridges106 which extend both into the plane of the Figure and parallel to theplane of the Figure. The ridges are preferably about 50 to 200 μm deepand spaced at about 2 to 3 mm. The purpose of the ridges is to generateturbulent flow for better mixing. The bottom surface of the cavity ispreferably light absorbing so as to prevent reflection of impinginglight.

A substrate 112 is mounted above the cavity 104. The substrate isprovided along its bottom surface 114 with a photoremovable protectivegroup such as NVOC with or without an intervening linker molecule. Thesubstrate is preferably transparent to a wide spectrum of light, but insome embodiments is transparent only at a wavelength at which theprotective group may be removed (such as UV in the case of NVOC). Thesubstrate in some embodiments is a conventional microscope glass slideor cover slip. The substrate is preferably as thin as possible, whilestill providing adequate physical support. Preferably, the substrate isless than about 1 mm thick, more preferably less than 0.5 mm thick, morepreferably less than 0.1 mm thick, and most preferably less than 0.05 mmthick. In alternative preferred embodiments, the substrate is quartz orsilicon.

The substrate and the body serve to seal the cavity except for an inletport 108 and an outlet port 110. The body and the substrate may be matedfor sealing in some embodiments with one or more gaskets. According to apreferred embodiment, the body is provided with two concentric gasketsand the intervening space is held at vacuum to ensure mating of thesubstrate to the gaskets.

Fluid is pumped through the inlet port into the cavity by way of a pump116 which may be, for example, a model no. B-120-S made by EldexLaboratories. Selected fluids are circulated into the cavity by thepump, through the cavity, and out the outlet for recirculation ordisposal. The reactor may be subjected to ultrasonic radiation and/orheated to aid in agitation in some embodiments.

Above the substrate 112, a lens 120 is provided which may be, forexample, a 2"100 mm focal length fused silica lens. For the sake of acompact system, a reflective mirror 122 may be provided for directinglight from a light source 124 onto the substrate. Light source 124 maybe, for example, a Xe(Hg) light source manufactured by Oriel and havingmodel no. 66024. A second lens 126 may be provided for the purpose ofprojecting a mask image onto the substrate in combination with lens 120.This form of lithography is referred to herein as projection printing.As will be apparent from this disclosure, proximity printing and thelike may also be used according to some embodiments.

Light from the light source is permitted to reach only selectedlocations on the substrate as a result of mask 128. Mask 128 may be, forexample, a glass slide having etched chrome thereon. The mask 128 in oneembodiment is provided with a grid of transparent locations and opaquelocations. Such masks may be manufactured by, for example, PhotoSciences, Inc. Light passes freely through the transparent regions ofthe mask, but is reflected from or absorbed by other regions. Therefore,only selected regions of the substrate are exposed to light.

As discussed above, light valves (LCD's) may be used as an alternativeto conventional masks to selectively expose regions of the substrate.Alternatively, fiber optic faceplates such as those available fromSchott Glass, Inc, may be used for the purpose of contrast enhancementof the mask or as the sole means of restricting the region to whichlight is applied. Such faceplates would be placed directly above or onthe substrate in the reactor shown in FIG. 8A. In still furtherembodiments, flys-eye lenses, tapered fiber optic faceplates, or thelike, may be used for contrast enhancement.

In order to provide for illumination of regions smaller than awavelength of light, more elaborate techniques may be utilized. Forexample, according to one preferred embodiment, light is directed at thesubstrate by way of molecular microcrystals on the tip of, for example,micropipettes. Such devices are disclosed in Lieberman et al., "A LightSource Smaller Than the Optical Wavelength," Science (1990) 247:59-61,which is incorporated herein by reference for all purposes.

In operation, the substrate is placed on the cavity and sealed thereto.All operations in the process of preparing the substrate are carried outin a room lit primarily or entirely by light of a wavelength outside ofthe light range at which the protective group is removed. For example,in the case of NVOC, the room should be lit with a conventional darkroom light which provides little or no UV light. All operations arepreferably conducted at about room temperature.

A first, deprotection fluid (without a monomer) is circulated throughthe cavity. The solution preferably is of 5 mM sulfuric acid in dioxanesolution which serves to keep exposed amino groups protonated anddecreases their reactivity with photolysis by-products. Absorptivematerials such as N,N-diethylamino 2,4-dinitrobenzene, for example, maybe included in the deprotection fluid which serves to absorb light andprevent reflection and unwanted photolysis.

The slide is, thereafter, positioned in a light raypath from the masksuch that first locations on the substrate are illuminated and,therefore, deprotected. In preferred embodiments the substrate isilluminated for between about 1 and 15 minutes with a preferredillumination time of about 10 minutes at 10-20 mW/cm² with 365 nm light.The slides are neutralized (i.e., brought to a pH of about 7) afterphotolysis with, for example, a solution of di-isopropylethylamine(DIEA) in methylene chloride for about 5 minutes.

The first monomer is then placed at the first locations on thesubstrate. After irradiation, the slide is removed, treated in bulk, andthen reinstalled in the flow cell. Alternatively, a fluid containing thefirst monomer, preferably also protected by a protective group, iscirculated through the cavity by way of pump 116. If, for example, it isdesired to attach the amino acid Y to the substrate at the firstlocations, the amino acid Y (bearing a protective group on itsα-nitrogen), along with reagents used to render the monomer reactive,and/or a carrier, is circulated from a storage container 118, throughthe pump, through the cavity, and back to the inlet of the pump.

The monomer carrier solution is, in a preferred embodiment, formed bymixing of a first solution (referred to herein as solution "A") and asecond solution (referred to herein as solution "B"). Table 2 providesan illustration of a mixture which may be used for solution A.

                  TABLE 2                                                         ______________________________________                                        Representative Monomer Carrier Solution "A"                                   ______________________________________                                        100 mg NVOC amino protected amino acid                                        37 mg HOBT (1-Hydroxybenzotriazole)                                           250 μl DMF (Dimethylformamide)                                             86 μl DIEA (Diisopropylethylamine)                                         ______________________________________                                    

The composition of solution B is illustrated in Table 3. Solutions A andB are mixed and allowed to react at room temperature for about 8minutes, then diluted with 2 ml of DMF, and 500 μl are applied to thesurface of the slide or the solution is circulated through the reactorsystem and allowed to react for about 2 hours at room temperature. Theslide is then washed with DMF, methylene chloride and ethanol.

                  TABLE 3                                                         ______________________________________                                        Representative Monomer Carrier Solution "B"                                   ______________________________________                                        250 μl DMF                                                                 111 mg BOP (Benzotriazolyl-n-oxy-tris(dimethylamino)                          phosphoniumhexafluorophosphate)                                               ______________________________________                                    

As the solution containing the monomer to be attached is circulatedthrough the cavity, the amino acid or other monomer will react at itscarboxy terminus with amino groups on the regions of the substrate whichhave been deprotected. Of course, while the invention is illustrated byway of circulation of the monomer through the cavity, the inventioncould be practiced by way of removing the slide from the reactor andsubmersing it in an appropriate monomer solution.

After addition of the first monomer, the solution containing the firstamino acid is then purged from the system. After circulation of asufficient amount of the DMF/methylene chloride such that removal of theamino acid can be assured (e.g., about 50×times the volume of the cavityand carrier lines), the mask or substrate is repositioned, or a new maskis utilized such that second regions on the substrate will be exposed tolight and the light 124 is engaged for a second exposure. This willdeprotect second regions on the substrate and the process is repeateduntil the desired polymer sequences have been synthesized.

The entire derivatized substrate is then exposed to a receptor ofinterest, preferably labeled with, for example, a fluorescent marker, bycirculation of a solution or suspension of the receptor through thecavity or by contacting the surface of the slide in bulk. The receptorwill preferentially bind to certain regions of the substrate whichcontain complementary sequences.

Antibodies are typically suspended in what is commonly referred to as"supercocktail," which may be, for example, a solution of about 1% BSA(bovine serum albumin), 0.5% Tween™ non-ionic detergent in PBS(phosphate buffered saline) buffer. The antibodies are diluted into thesupercocktail buffer to a final concentration of, for example, about 0.1to 4 μg/ml.

FIG. 8B illustrates an alternative preferred embodiment of the reactorshown in FIG. 8A. According to this embodiment, the mask 128 is placeddirectly in contact with the substrate. Preferably, the etched portionof the mask is placed face down so as to reduce the effects of lightdispersion. According to this embodiment, the imaging lenses 120 and 126are not necessary because the mask is brought into close proximity withthe substrate.

For purposes of increasing the signal-to-noise ratio of the technique,some embodiments of the invention provide for exposure of the substrateto a first labeled or unlabeled receptor followed by exposure of alabeled, second receptor (e.g., an antibody) which binds at multiplesites on the first receptor. If, for example, the first receptor is anantibody derived from a first species of an animal, the second receptoris an antibody derived from a second species directed to epitopesassociated with the first species. In the case of a mouse antibody, forexample, fluorescently labeled goat antibody or antiserum which isantimouse may be used to bind at multiple sites on the mouse antibody,providing several times the fluorescence compared to the attachment of asingle mouse antibody at each binding site. This process may be repeatedagain with additional antibodies (e.g., goat-mouse-goat, etc.) forfurther signal amplification.

In preferred embodiments an ordered sequence of masks is utilized. Insome embodiments it is possible to use as few as a single mask tosynthesize all of the possible polymers of a given monomer set.

If, for example, it is desired to synthesize all 16 dinucleotides fromfour bases, a 1 cm square synthesis region is divided conceptually into16 boxes, each 0.25 cm wide. Denote the four monomer units by A, B, C,and D. The first reactions are carried out in four vertical columns,each 0.25 cm wide. The first mask exposes the left-most column of boxes,where A is coupled. The second mask exposes the next column, where B iscoupled; followed by a third mask, for the C column; and a final maskthat exposes the right-most column, for D. The first, second, third, andfourth masks may be a single mask translated to different locations.

The process is repeated in the horizontal direction for the second unitof the dimer. This time, the masks allow exposure of horizontal rows,again 0.25 cm wide. A, B, C, and D are sequentially coupled using masksthat expose horizontal fourths of the reaction area. The resultingsubstrate contains all 16 dinucleotides of four bases.

The eight masks used to synthesize the dinucleotide are related to oneanother by translation or rotation. In fact, one mask can be used in alleight steps if it is suitably rotated and translated. For example, inthe example above, a mask with a single transparent region could besequentially used to expose each of the vertical columns, translated90°, and then sequentially used to allow exposure of the horizontalrows.

Tables 4 and 5 provide a simple computer program in Quick Basic forplanning a masking program and a sample output, respectively, for thesynthesis of a polymer chain of three monomers ("residues") having threedifferent monomers in the first level, four different monomers in thesecond level, and five different monomers in the third level in astriped pattern. The output of the program is the number of cells, thenumber of "stripes" (light regions) on each mask, and the amount oftranslation required for each exposure of the mask.

                                      TABLE 4                                     __________________________________________________________________________    Mask Strategy Program                                                         __________________________________________________________________________    DEFINT A-Z                                                                    DIM b(20), w(20), 1(500)                                                      F$ = "LPT1:"                                                                  OPEN f$ FOR OUTPUT AS #1                                                      jmax = 3                                                                              `Number of residues                                                   b(1) = 3: b(2) = 4: b(3) = 5                                                                 `Number of building blocks for res 1,2,3                       g = 1: 1max(1) = 1                                                            FOR j = 1 TO jmax: g= g * b(j): NEXT j                                        w(0) = 0: w(1) = g / b(1)                                                     PRINT #1, "MASK2.BAS ", DATE$, TIME$: PRINT #1,                               PRINT #1, USING "Number of residues=##"; jmax                                 FOR j = 1 TO jmax                                                             PRINT #1, USING "  Residue ##  ## building blocks"; j; b(j)                   NEXT j                                                                        PRINT #1, "                                                                   PRINT #1, USING "Number of cells=####"; g: PRINT #1,                          FOR j = 2 TO jmax                                                             1max(j) =  1max(j - 1) * b(j - 1)                                             w(j) = w(j - 1) / b(j)                                                        NEXT j                                                                        FOR j = 1 TO jmax                                                             PRINT #1, USING "Mask for residue ##"; j: PRINT #1,                           PRINT #1, USING " Number of stripes=###"; 1max(j)                             PRINT #1, USING " Width of each stripe=###"; w(j)                             FOR 1 = 1 TO 1max(j)                                                          a = 1 + (1 - 1) * w(j - 1)                                                    ae = a + w(j) - 1                                                             PRINT #1, USING " Stripe ## begins at location ### and ends at ###"; 1;       a; ae                                                                         NEXT 1                                                                        PRINT #1,                                                                     PRINT #1, USING " For each of ## building blocks, translate mask by ##        cell(s)"; b(j); w(j),                                                         PRINT #1, : PRINT #1, : PRINT #1,                                             NEXT j                                                                        __________________________________________________________________________     © Copyright 1990, Affymax Research Institute                     

                  TABLE 5                                                         ______________________________________                                        Masking Strategy Output                                                       ______________________________________                                        Number of residues= 3                                                         Residue 1   3 building blocks                                                 Residue 2   4 building blocks                                                 Residue 3   5 building blocks                                                 Number of cells= 60                                                           Mask for residue 1                                                            Number of stripes= 1                                                          Width of each stripe= 20                                                      Stripe 1 begins at location 1 and ends at 20                                  For each of 3 building blocks, translate mask by 20 cell(s)                   Mask for residue 2                                                            Number of stripes= 3                                                          Width of each stripe= 5                                                       Stripe 1 begins at location 1 and ends at 5                                   Stripe 2 begins at location 21 and ends at 25                                 Stripe 3 begins at location 41 and ends at 45                                 For each of 4 building blocks, translate mask by 5 cell(s)                    Mask for residue 3                                                            Number of stripes= 12                                                         Width of each stripe= 1                                                       Stripe 1 begins at location 1 and ends at 1                                   Stripe 2 begins at location 6 and ends at 6                                   Stripe 3 begins at location 11 and ends at 11                                 Stripe 4 begins at location 16 and ends at 16                                 Stripe 5 begins at location 21 and ends at 21                                 Stripe 6 begins at location 26 and ends at 26                                 Stripe 7 begins at location 31 and ends at 31                                 Stripe 8 begins at location 36 and ends at 36                                 Stripe 9 begins at location 41 and ends at 41                                 Stripe 10 begins at location 46 and ends at 46                                Stripe 11 begins at location 51 and ends at 51                                Stripe 12 begins at location 56 and ends at 56                                For each of 5 building blocks, translate mask by 1 cell(s)                    ______________________________________                                         © Copyright 1990, Affymax Research Institute                     

V. Details of One Embodiment of A Fluorescent Detection Device

FIG. 9 illustrates a fluorescent detection device for detectingfluorescently labeled receptors on a substrate. A substrate 112 isplaced on an x/y translation table 202. In a preferred embodiment thex/y translation table is a model no. PM500-A1 manufactured by NewportCorporation. The x/y translation table is connected to and controlled byan appropriately programmed digital computer 204 which may be, forexample, an appropriately programmed IBM PC/AT or AT compatiblecomputer. Of course, other computer systems, special purpose hardware,or the like could readily be substituted for the AT computer used hereinfor illustration. Computer software for the translation and datacollection functions described herein can be provided based oncommercially available software including, for example, "Lab Windows"licensed by National Instruments, which is incorporated herein byreference for all purposes.

The substrate and x/y translation table are placed under a microscope206 which includes one or more objectives 208. Light (about 488 nm) froma laser 210, which in some embodiments is a model no. 2020-05 argon ionlaser manufactured by Spectraphysics, is directed at the substrate by adichroic mirror 207 which passes greater than about 520 nm light butreflects 488 nm light. Dichroic mirror 207 may be, for example, a modelno. FT510 manufactured by Carl Zeiss. Light reflected from the mirrorthen enters the microscope 206 which may be, for example, a model no.Axioscop 20 manufactured by Carl Zeiss. Fluorescein-marked materials onthe substrate will fluoresce >488 nm light, and the fluoresced lightwill be collected by the microscope and passed through the mirror. Thefluorescent light from the substrate is then directed through awavelength filter 209 and, thereafter through an aperture plate 211.Wavelength filter 209 may be, for example, a model no. OG530manufactured by Melles Griot and aperture plate 211 may be, for example,a model no. 477352/477380 manufactured by Carl Zeiss.

The fluoresced light then enters a photomultiplier tube 212 which insome embodiments is a model no. R943-02 manufactured by Hamamatsu, thesignal is amplified in preamplifier 214 and photons are counted byphoton counter 216. The number of photons is recorded as a function ofthe location in the computer 204. Pre-Amp 214 may be, for example, amodel no. SR440 manufactured by Stanford Research Systems and photoncounter 216 may be a model no. SR400 manufactured by Stanford ResearchSystems. The substrate is then moved to a subsequent location and theprocess is repeated. In preferred embodiments the data are acquiredevery 1 to 100 μm with a data collection diameter of about 0.8 to 10 μmpreferred. In embodiments with sufficiently high fluorescence, a CCD(change coupled device) detector with broadfield illumination isutilized.

By counting the number of photons generated in a given area in responseto the laser, it is possible to determine where fluorescent markedmolecules are located on the substrate. Consequently, for a slide whichhas a matrix of polypeptides, for example, synthesized on the surfacethereof, it is possible to determine which of the polypeptides iscomplementary to a fluorescently marked receptor.

According to preferred embodiments, the intensity and duration of thelight applied to the substrate is controlled by varying the laser powerand scan stage rate for improved signal-to-noise ratio by maximizingfluorescence emission and minimizing background noise.

While the detection apparatus has been illustrated primarily herein withregard to the detection of marked receptors, the invention will findapplication in other areas. For example, the detection apparatusdisclosed herein could be used in the fields of catalysis, DNA orprotein gel scanning, and the like.

VI. Determination of Relative Binding Strength of Receptors

The signal-to-noise ratio of the present invention is sufficiently highthat not only can the presence or absence of a receptor on a ligand bedetected, but also the relative binding affinity of receptors to avariety of sequences can be determined.

In practice it is found that a receptor will bind to several peptidesequences in an array, but will bind much more strongly to somesequences than others. Strong binding affinity will be evidenced hereinby a strong fluorescent or radiographic signal since many receptormolecules will bind in a region of a strongly bound ligand. Conversely,a weak binding affinity will be evidenced by a weak fluorescent orradiographic signal due to the relatively small number of receptormolecules which bind in a particular region of a substrate having aligand with a weak binding affinity for the receptor. Consequently, itbecomes possible to determine relative binding avidity (or affinity inthe case of univalent interactions) of a ligand herein by way of theintensity of a fluorescent or radiographic signal in a region containingthat ligand.

Semiquantitative data on affinities might also be obtained by varyingwashing conditions and concentrations of the receptor. This would bedone by comparison to known ligand receptor pairs, for example.

VII. Examples

The following examples are provided to illustrate the efficacy of theinventions herein. All operations were conducted at about ambienttemperatures and pressures unless indicated to the contrary.

A. Slide Preparation

Before attachment of reactive groups it is preferred to clean thesubstrate which is, in a preferred embodiment a glass substrate such asa microscope slide or cover slip. According to one embodiment the slideis soaked in an alkaline bath consisting of, for example, 1 liter of 95%ethanol with 120 ml of water and 120 grams of sodium hydroxide for 12hours. The slides are then washed under running water and allowed to airdry, and rinsed once with a solution of 95% ethanol.

The slides are then aminated with, for example,aminopropyltriethoxysilane for the purpose of attaching amino groups tothe glass surface on linker molecules, although any omega functionalizedsilane could also be used for this purpose. In one embodiment 0.1%aminopropyltriethoxysilane is utilized, although solutions withconcentrations from 10⁻⁷ % to 10% may be used, with about 10⁻³ % to 2%preferred. A 0.1% mixture is prepared by adding to 100 ml of a 95%ethanol/5% water mixture, 100 microliters (μl) ofaminopropyltriethoxysilane. The mixture is agitated at about ambienttemperature on a rotary shaker for about 5 minutes. 500 μl of thismixture is then applied to the surface of one side of each cleanedslide. After 4 minutes, the slides are decanted of this solution andrinsed three times by dipping in, for example, 100% ethanol.

After the plates dry, they are placed in a 110°-120° C. vacuum oven forabout 20 minutes, and then allowed to cure at room temperature for about12 hours in an argon environment. The slides are then dipped into DMF(dimethylformamide) solution, followed by a thorough washing withmethylene chloride.

The aminated surface of the slide is then exposed to about 500 μl of,for example, a 30 millimolar (mM) solution of NVOC-GABA (gamma aminobutyric acid) NHS (N-hydroxysuccinimide) in DMF for attachment of aNVOC-GABA to each of the amino groups.

The surface is washed with, for example, DMF, methylene chloride, andethanol.

Any unreacted aminopropyl silane on the surface--that is, those aminogroups which have not had the NVOC-GABA attached--are now capped withacetyl groups (to prevent further reaction) by exposure to a 1:3 mixtureof acetic anhydride in pyridine for 1 hour. Other materials which mayperform this residual capping function include trifluoroaceticanhydride, formicacetic anhydride, or other reactive acylating agents.Finally, the slides are washed again with DMF, methylene chloride, andethanol.

B. Synthesis of Eight Trimers of "A" and "B"

FIG. 10 illustrates a possible synthesis of the eight trimers of thetwo-monomer set: gly, phe (represented by "A" and "B," respectively). Aglass slide bearing silane groups terminating in6-nitroveratryloxycarboxamide (NVOC-NH) residues is prepared as asubstrate. Active esters (pentafluorophenyl, OBt, etc.) of gly and pheprotected at the amino group with NVOC are prepared as reagents. Whilenot pertinent to this example, if side chain protecting groups arerequired for the monomer set, these must not be photoreactive at thewavelength of light used to protect the primary chain.

For a monomer set of size n, n×l cycles are required to synthesize allpossible sequences of length l. A cycle consists of:

1. Irradiation through an appropriate mask to expose the amino groups atthe sites where the next residue is to be added, with appropriate washesto remove the by-products of the deprotection.

2. Addition of a single activated and protected (with the samephotochemically-removable group) monomer, which will react only at thesites addressed in step 1, with appropriate washes to remove the excessreagent from the surface.

The above cycle is repeated for each member of the monomer set untileach location on the surface has been extended by one residue in oneembodiment. In other embodiments, several residues are sequentiallyadded at one location before moving on to the next location. Cycle timeswill generally be limited by the coupling reaction rate, now as short as20 min in automated peptide synthesizers. This step is optionallyfollowed by addition of a protecting group to stabilize the array forlater testing. For some types of polymers (e.g., peptides), a finaldeprotection of the entire surface (removal of photoprotective sidechain groups) may be required.

More particularly, as shown in FIG. 10A, the glass 20 is provided withregions 22, 24, 26, 28, 30, 32, 34, and 36. Regions 30, 32, 34, and 36are masked, as shown in FIG. 10B and the glass is irradiated and exposedto a reagent containg "A" (e.g., gly), with the resulting structureshown in FIG. 10C. Thereafter, regions 22, 24, 26, and 28 are masked,the glass is irradiated (as shown in FIG. 10D) and exposed to a reagentcontaining "B" (e.g., phe), with the resulting structure shown in FIG.10E. The process proceeds, consecutively masking and exposing thesections as shown until the structure shown in FIG. 10M is obtained. Theglass is irradiated and the terminal groups are, optionally, capped byacetylation. As shown, all possible trimers of gly/phe are obtained.

In this example, no side chain protective group removal is necessary. Ifit is desired, side chain deprotection may be accomplished by treatmentwith ethanedithiol and trifluoroacetic acid.

In general, the number of steps needed to obtain a particular polymerchain is defined by:

    n×l                                                  (1)

where:

n=the number of monomers in the basis set of monomers, and

l=the number of monomer units in a polymer chain.

Conversely, the synthesized number of sequences of length l will be:

    n.sup.l.                                                   (2)

Of course, greater diversity is obtained by using masking strategieswhich will also include the synthesis of polymers having a length ofless than l. If, in the extreme case, all polymers having a length lessthan or equal to l are synthesized, the number of polymers synthesizedwill be:

    n.sup.l +n.sup.l-1 + . . . +n.sup.1.                       (3)

The maximum number of lithographic steps needed will generally be n foreach "layer" of monomers, i.e., the total number of masks (and,therefore, the number of lithographic steps) needed will be n×l. Thesize of the transparent mask regions will vary in accordance with thearea of the substrate available for synthesis and the number ofsequences to be formed. In general, the size of the synthesis areas willbe:

    size of synthesis areas=(A)/(Sequences)

where:

A is the total area available for synthesis; and

Sequences is the number of sequences desired in the area.

It will be appreciated by those of skill in the art that the abovemethod could readily be used to simultaneously produce thousands ormillions of oligomers on a substrate using the photolithographictechniques disclosed herein. Consequently, the method results in theability to practically test large numbers of, for example, di, tri,tetra, penta, hexa, hepta, octapeptides, dodecapeptides, or largerpolypeptides (or correspondingly, polynucleotides).

The above example has illustrated the method by way of a manual example.It will of course be appreciated that automated or semi-automatedmethods could be used. The substrate would be mounted in a flow cell forautomated addition and removal of reagents, to minimize the volume ofreagents needed, and to more carefully control reaction conditions.Successive masks could be applied manually or automatically.

Synthesis of a Dimer of an Aminopropyl Group and a Fluorescent Group

In synthesizing the dimer of an aminopropyl group and a fluorescentgroup, a functionalized durapore membrane was used as a substrate. Thedurapore membrane was a polyvinylidine difluoride with aminopropylgroups. The aminopropyl groups were protected with the DDZ group byreaction of the carbonyl chloride with the amino groups, a reactionreadily known to those of skill in the art. The surface bearing thesegroups was placed in a solution of THF and contacted with a mask bearinga checkerboard pattern of 1 mm opaque and transparent regions. The maskwas exposed to ultraviolet light having a wavelength down to at leastabout 280 nm for about 5 minutes at ambient temperature, although a widerange of exposure times and temperatures may be appropriate in variousembodiments of the invention. For example, in one embodiment, anexposure time of between about 1 and 5000 seconds may be used at processtemperatures of between -70° and +50° C.

In one preferred embodiment, exposure times of between about 1 and 500seconds at about ambient pressure are used. In some preferredembodiments, pressure above ambient is used to prevent evaporation.

The surface of the membrane was then washed for about 1 hour with afluorescent label which included an active ester bound to a chelate of alanthanide. Wash times will vary over a wide range of values from abouta few minutes to a few hours. These materials fluoresce in the red andthe green visible region. After the reaction with the active ester inthe fluorophore was complete, the locations in which the fluorophore wasbound could be visualized by exposing them to ultraviolet light andobserving the red and the green fluorescence. It was observed that thederivatized regions of the substrate closely corresponded to theoriginal pattern of the mask.

D. Demonstration of Signal Capability

Signal detection capability was demonstrated using a low-level standardfluorescent bead kit manufactured by Flow Cytometry Standards and havingmodel no. 824. This kit includes 5.8 μm diameter beads, each impregnatedwith a known number of fluorescein molecules.

One of the beads was placed in the illumination field on the scan stageas shown in FIG. 9 in a field of a laser spot which was initiallyshuttered. After being positioned in the illumination field, the photondetection equipment was turned on. The laser beam was unblocked and itinteracted with the particle bead, which then fluoresced. Fluorescencecurves of beads impregnated with 7,000 and 13,000; fluoresceinmolecules, are shown in FIGS. 11A and 11B respectively. On each curve,traces for beads without fluorescein molecules are also shown. Theseexperiments were performed with 488 nm excitation, with 100 μW of laserpower. The light was focused through a 40 power 0.75 NA objective.

The fluorescence intensity in all cases started off at a high value andthen decreased exponentially. The fall-off in intensity is due tophotobleaching of the fluorescein molecules. The traces of beads withoutfluorescein molecules are used for background subtraction. Thedifference in the initial exponential decay between labeled andnonlabeled beads is integrated to give the total number of photoncounts, and this number is related to the number of molecules per bead.Therefore, it is possible to deduce the number of photons perfluorescein molecule that can be detected. For the curves illustrated inFIG. 11A and 11B, this calculation indicates the radiation of about 40to 50 photons per fluorescein molecule are detected.

E. Determination of the Number of

Molecules Per Unit Area

Aminopropylated glass microscope slides prepared according to themethods discussed above were utilized in order to establish the densityof labeling of the slides. The free amino termini of the slides werereacted with FITC (fluorescein isothiocyanate) which forms a covalentlinkage with the amino group. The slide is then scanned to count thenumber of fluorescent photons generated in a region which, using theestimated 40-50 photons per fluorescent molecule, enables thecalculation of the number of molecules which are on the surface per unitarea.

A slide with aminopropyl silane on its surface was immersed in a 1 mMsolution of FITC in DMF for 1 hour at about ambient temperature. Afterreaction, the slide was washed twice with DMF and then washed withethanol, water, and then ethanol again. It was then dried and stored inthe dark until it was ready to be examined.

Through the use of curves similar to those shown in FIG. 11A and 11B,and by integrating the fluorescent counts under the exponentiallydecaying signal, the number of free amino groups on the surface afterderivatization was determined. It was determined that slides withlabeling densities of 1 fluorescein per 10³ ×10³ to ˜2×2 nm could bereproducibly made as the concentration of aminopropyltriethoxysilanevaried from 10⁻⁵ % to 10⁻¹ %.

F. Removal of NVOC and Attachment of A Fluorescent Marker

NVOC-GABA groups were attached as described above. The entire surface ofone slide was exposed to light so as to expose a free amino group at theend of the gamma amino butyric acid. This slide, and a duplicate whichwas not exposed, were then exposed to fluorescein isothiocyanate (FITC).

FIG. 12A illustrates the slide which was not exposed to light, but whichwas exposed to FITC. The units of the x axis are time and the units ofthe y axis are counts. The trace contains a certain amount of backgroundfluorescence. The duplicate slide was exposed to 350 nm broadbandillumination for about 1 minute (12 mW/cm², ˜350 nm illumination),washed and reacted with FITC. The fluorescence curves for this slide areshown in FIG. 12B. A large increase in the level of fluorescence isobserved, which indicates photolysis has exposed a number of aminogroups on the surface of the slides for attachment of a fluorescentmarker.

G. Use of a Mask in Removal of NVOC

The next experiment was performed with a 0.1% aminopropylated slide.Light from a Hg--Xe arc lamp was imaged onto the substrate through alaser-ablated chrome-on-glass mask in direct contact with the substrate.

This slide was illuminated for approximately 5 minutes, with 12 mW of350 nm broadband light and then reacted with the 1 mM FITC solution. Itwas put on the laser detection scanning stage and a graph was plotted asa two-dimensional representation of position color-coded forfluorescence intensity. The fluorescence intensity (in counts) as afunction of location is given on the color scale to the right of FIG.13A for a mask having 100×100 μm squares.

The experiment was repeated a number of times through various masks. Thefluorescence pattern for a 50 μm mask is illustrated in FIG. 13B, for a20 μm mask in FIG. 13C, and for a 10 μm mask in FIG. 13D. The maskpattern is distinct down to at least about 10 μm squares using thislithographic technique.

H. Attachment of YGGFL and Subsequent Exposure to

Herz Antibody and Goat Antimouse

In order to establish that receptors to a particular polypeptidesequence would bind to a surface-bound peptide and be detected, Leuenkephalin was coupled to the surface and recognized by an antibody. Aslide was derivatized with 0.1% amino propyl-triethoxysilane andprotected with NVOC. A 500 μm checkerboard mask was used to expose theslide in a flow cell using backside contact printing. The Leu enkephalinsequence (H₂ N-tyrosine,glycine,glycine,phenylalanine,leucine-CO₂ H,otherwise referred to herein as YGGFL) was attached via its carboxy endto the exposed amino groups on the surface of the slide. The peptide wasadded in DMF solution with the BOP/HOBT/DIEA coupling reagents andrecirculated through the flow cell for 2 hours at room temperature.

A first antibody, known as the Herz antibody, was applied to the surfaceof the slide for 45 minutes at 2 μg/ml in a supercocktail (containing 1%BSA and 1% ovalbumin also in this case). A second antibody, goatanti-mouse fluorescein conjugate, was then added at 2 μg/ml in thesupercocktail buffer, and allowed to incubate for 2 hours. An imagetaken at 10 μm steps indicated that not only can deprotection be carriedout in a well defined pattern, but also that (1) the method provides forsuccessful coupling of peptides to the surface of the substrate, (2) thesurface of a bound peptide is available for binding with an antibody,and (3) that the detection apparatus capabilities are sufficient todetect binding of a receptor.

I. Monomer-by-Monomer Formation of YGGFL and Subsequent Exposure toLabeled Antibody

Monomer-by-monomer synthesis of YGGFL and GGFL in alternate squares wasperformed on a slide in a checkerboard pattern and the resulting slidewas exposed to the Herz antibody. This experiment and the resultsthereof are illustrated in FIGS. 14A, 14B, 15A, and 15B.

In FIG. 14A, a slide is shown which is derivatized with the aminopropylgroup, protected in this case with t-BOC (t-butoxycarbonyl). The slidewas treated with TFA to remove the t-BOC protecting group.E-aminocaproic acid, which was t-BOC protected at its amino group, wasthen coupled onto the aminopropyl groups. The aminocaproic acid servesas a spacer between the aminopropyl group and the peptide to besynthesized. The amino end of the spacer was deprotected and coupled toNVOC-leucine. The entire slide was then illuminated with 12 mW of 325 nmbroadband illumination. The slide was then coupled withNVOC-phenylalanine and washed. The entire slide was again illuminated,then coupled to NVOC-glycine and washed. The slide was again illuminatedand coupled to NVOC-glycine to form the sequence shown in the lastportion of FIG. 14A.

As shown in FIG. 14B, alternating regions of the slide were thenilluminated using a projection print using a 500×500 μm checkerboardmask; thus, the amino group of glycine was exposed only in the lightedareas. When the next coupling chemistry step was carried out,NVOC-tyrosine was added, and it coupled only at those spots which hadreceived illumination. The entire slide was then illuminated to removeall the NVOC groups, leaving a checkerboard of YGGFL in the lightedareas and in the other areas, GGFL. The Herz antibody (which recognizesthe YGGFL, but not GGFL) was then added, followed by goat anti-mousefluorescein conjugate.

The resulting fluorescence scan is shown in FIG. 15A, and the colorcoding for the fluorescence intensity is again given on the right. Darkareas contain the tetrapeptide GGFL, which is not recognized by the Herzantibody (and thus there is no binding of the goat anti-mouse antibodywith fluorescein conjugate), and in the red areas YGGFL is present. TheYGGFL pentapeptide is recognized by the Herz antibody and, therefore,there is antibody in the lighted regions for the fluorescein-conjugatedgoat anti-mouse to recognize.

Similar patterns are shown for a 50 μm mask used in direct contact("proximity print") with the substrate in FIG. 15B. Note that thepattern is more distinct and the corners of the checkerboard pattern aretouching when the mask is placed in direct contact with the substrate(which reflects the increase in resolution using this technique).

J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL

A synthesis using a 50 μm checkerboard mask similar to that shown inFIG. 15B was conducted. However, P was added to the GGFL sites on thesubstrate through an additional coupling step. P was added by exposingprotected GGFL to light and subsequent exposure to P in the manner setforth above. Therefore, half of the regions on the substrate containedYGGFL and the remaining half contained PGGFL.

The fluorescence plot for this experiment is provided in FIG. 16. Asshown, the regions are again readily discernable. This experimentdemonstrates that antibodies are able to recognize a specific sequenceand that the recognition is not length-dependent.

K. Monomer-by-Monomer Synthesis of YGGFL and YPGGFL

In order to further demonstrate the operability of the invention, a 50μm checkerboard pattern of alternating YGGFL and YPGGFL was synthesizedon a substrate using techniques like those set forth above. Theresulting fluorescence plot is provided in FIG. 17. Again, it is seenthat the antibody is clearly able to recognize the YGGFL sequence anddoes not bind significantly at the YPGGFL regions.

L. Synthesis of an Array of Sixteen Different Amino Acid Sequences andEstimation of Relative Binding Affinity to Herz Antibody

Using techniques similar to those set forth above, an array of 16different amino acid sequences (replicated four times) was synthesizedon each of two glass substrates. The sequences were synthesized byattaching the sequence NVOC-GFL across the entire surface of the slides.Using a series of masks, two layers of amino acids were then selectivelyapplied to the substrate. Each region had dimensions of 0.25 cm×0.0625cm. The first slide contained amino acid sequences containing only Lamino acids while the second slide contained selected D amino acids.FIGS. 18A and 18B illustrate a map of the various regions on the firstand second slides, respectively. The patterns shown in FIGS. 18A and 18Bwere duplicated four times on each slide. The slides were then exposedto the Herz antibody and fluorescein-labeled goat anti-mouse.

FIG. 19 is a fluorescence plot of the first slide, which contained onlyL amino acids. Red indicates strong binding (149,000 counts or more)while black indicates little or no binding of the Herz antibody (20,000counts or less). The bottom right-hand portion of the slide appears "cutoff" because the slide was broken during processing. The sequence YGGFLis clearly most strongly recognized. The sequences YAGFL and YSGFL alsoexhibit strong recognition of the antibody. By contrast, most of theremaining sequences show little or no binding. The four duplicateportions of the slide are extremely consistent in the amount of bindingshown therein.

FIG. 20 is a fluorescence plot of the second slide. Again, strongestbinding is exhibited by the YGGFL sequence. Significant binding is alsodetected to YaGFL, YsGFL, and YpGFL (where L-amino acids are identifiedby one upper case letter abbreviation, and D-amino acids are identifiedby one lower case letter abbreviation). The remaining sequences showless binding with the antibody. Note the low binding efficiency of thesequence yGGFL.

Table 6 lists the various sequences tested in order of relativefluorescence, which provides information regarding relative bindingaffinity.

                  TABLE 6                                                         ______________________________________                                        Apparent Binding to Herz Ab                                                          L-a.a. Set   D-a.a. Set                                                ______________________________________                                               YGGFL        YGGFL                                                            YAGFL        YaGFL                                                            YSGFL        YsGFL                                                            LGGFL        YpGFL                                                            FGGFL        fGGFL                                                            YPGFL        yGGFL                                                            LAGFL        faGFL                                                            FAGFL        wGGFL                                                            WGGFL        yaGFL                                                                         fpGFL                                                                         waGFL                                                     ______________________________________                                    

VIII. Illustrative Alternative Embodiment

According to an alternative embodiment of the invention, the methodsprovide for attaching to the surface a caged binding member which in itscaged form has a relatively low affinity for other potentially bindingspecies, such as receptors and specific binding substances.

According to this alternative embodiment, the invention provides methodsfor forming predefined regions on a surface of a solid support, whereinthe predefined regions are capable of immobilizing receptors. Themethods make use of caged binding members attached to the surface toenable selective activation of the predefined regions. The caged bindingmembers are liberated to act as binding members ultimately capable ofbinding receptors upon selective activation of the predefined regions.The activated binding members are then used to immobilize specificmolecules such as receptors on the predefined region of the surface. Theabove procedure is repeated at the same or different sites on thesurface so as to provide a surface prepared with a plurality of regionson the surface containing, for example, the same or different receptors.When receptors immobilized in this way have a differential affinity forone or more ligands, screenings and assays for the ligands can beconducted in the regions of the surface containing the receptors.

The alternative embodiment may make use of novel caged binding membersattached to the substrate. Caged (unactivated) members have a relativelylow affinity for receptors of substances that specifically bind touncaged binding members when compared with the corresponding affinitiesof activated binding members. Thus, the binding members are protectedfrom reaction until a suitable source of energy is applied to theregions of the surface desired to be activated. Upon application of asuitable energy source, the caging groups labilize, thereby presentingthe activated binding member. A typical energy source will be light.

Once the binding members on the surface are activated they may beattached to a receptor. The receptor chosen may be a monoclonalantibody, a nucleic acid sequence, a drug receptor, etc. The receptorwill usually, though not always, be prepared so as to permit attachingit, directly or indirectly, to a binding member. For example, a specificbinding substance having a strong binding affinity for the bindingmember and a strong affinity for the receptor or a conjugate of thereceptor may be used to act as a bridge between binding members andreceptors if desired. The method uses a receptor prepared such that thereceptor retains its activity toward a particular ligand.

Preferably, the caged binding member attached to the solid substratewill be a photoactivatable biotin complex, i.e., a biotin molecule thathas been chemically modified with photoactivatable protecting groups sothat it has a significantly reduced binding affinity for avidin oravidin analogs than does natural biotin. In a preferred embodiment, theprotecting groups localized in a predefined region of the surface willbe removed upon application of a suitable source of radiation to givebinding members, that are biotin or a functionally analogous compoundhaving substantially the same binding affinity for avidin or avidinanalogs as does biotin.

In another preferred embodiment, avidin or an avidin analog is incubatedwith activated binding members on the surface until the avidin bindsstrongly to the binding members. The avidin so immobilized on predefinedregions of the surface can then be incubated with a desired receptor orconjugate of a desired receptor. The receptor will preferably bebiotinylated, e.g., a biotinylated antibody, when avidin is immobilizedon the predefined regions of the surface. Alternatively, a preferredembodiment will present an avidin/biotinylated receptor complex, whichhas been previously prepared, to activated binding members on thesurface.

IX. Conclusion

The present inventions provide greatly improved methods and apparatusfor synthesis of polymers on substrates. It is to be understood that theabove description is intended to be illustrative and not restrictive.Many embodiments will be apparent to those of skill in the art uponreviewing the above description. By way of example, the invention hasbeen described primarily with reference to the use of photoremovableprotective groups, but it will be readily recognized by those of skillin the art that sources of radiation other than light could also beused. For example, in some embodiments it may be desirable to useprotective groups which are sensitive to electron beam irradiation,x-ray irradiation, in combination with electron beam lithograph, orx-ray lithography techniques. Alternatively, the group could be removedby exposure to an electric current. The scope of the invention should,therefore, be determined not with reference to the above description,but should instead be determined with reference to the appended claims,along with the full scope of equivalents to which such claims areentitled.

What is claimed is:
 1. A substrate with a surface comprising 10³ or moregroups of oligonucleotides with different, known sequences covalentlyattached to the surface in discrete known regions, said 10³ or moregroups of oligonucleotides occupying a total area of less than 1 cm² onsaid substrate, said groups of oligonucleotides having differentnucleotide sequences.
 2. The substrate as recited in claim 1 whereinsaid substrate comprises 10⁴ or more different groups of oligonucleotidewith known sequences covalently coupled to discrete known regions ofsaid substrate.
 3. The substrate as recited in claim 1 wherein saidsubstrate comprises 10⁵ or more different groups of oligonucleotideswith known sequences in discrete known regions.
 4. The substrate asrecited in claim 1 wherein said substrate comprises 10⁶ or moredifferent groups of oligonucleotides with known sequences in discreteknown regions.
 5. The substrate as recited in claim 1 wherein saidgroups of oligonucleotides are at least 50% pure within said discreteknown regions.
 6. The substrate as recited in claim 1 wherein the groupsof oligonucleotides are attached to the surface by a linker.
 7. An arrayof more than 1,000 different groups of oligonucleotide molecules withknown sequences covalently coupled to a surface of a substrate, saidgroups of oligonucleotide molecules each in discrete known regions anddiffering from other groups of oligonucleotide molecules in monomersequence, each of said discrete known regions being an area of less thanabout 0.01 cm² and each discrete known region comprisingoligonucleotides of known sequence, said different groups occupying atotal area of less than 1 cm².
 8. The array as recited in claim 7wherein said area is less than 10,000 microns².
 9. The array as recitedin claim 7 made by the process of:exposing a first region of saidsubstrate to light to remove photoremovable groups from nucleic acids insaid first region, and not exposing a second region of said surface tolight; covalently coupling a first nucleotide to said nucleic acids onsaid part of said substrate exposed to light, said first nucleotidecovalently coupled to said photoremovable group; exposing a part of saidfirst region of said substrate to light, and not exposing another partof said first region of said substrate to light to remove saidphotoremovable groups; covalently coupling a second nucleotide to saidpart of said first region exposed to light; and repeating said steps ofexposing said substrate to light and covalently coupling nucleotidesuntil said more than 500 different groups of nucleotides are formed onsaid surface.
 10. The array as recited in claim 7 comprising more than10,000 groups of oligonucleotides of known sequences.